MECHANISM OF NERVE GROWTH AND NAVIGATION

理解轴突寻路机制是神经科学的一个中心问题， 发育神经生物学本提案中概述的工作重点是 在分子和细胞机制的基础上， 神经过程的导航。我将使用的实验系统 将非洲爪蟾神经元的体外研究与最先进的成像技术相结合 分子操作技术在博士的实验室Mu-ming Poo。 本建议所涉及的主要问题是第二信使和 细胞骨架I蛋白参与指导生长锥运动。 这项建议有两个具体目标。 在AIM 1中，我将确定Ca 2+和cAMP作为第二信使的作用 在不同条件下调节神经突延伸的速度和方向 环境条件这将通过测量[Ca 2 +]i进行测定， 使用标准Fura-2比率成像方法的规则时间间隔，以及 通过记录明视野图像测量神经突延伸的速率 使用延时视频记录。无论是胞浆Ca 2+还是cAMP 梯度是否足以诱导生长锥转向，将通过 使用从笼状化合物中的Ca 2+和cAMP的焦点UV活化释放， 生长锥Ca 2+和cAMP依赖性通路是否与 也将互相检查。在AIM 2中，我将确定早期 与三种诱导的生长锥转向相关的细胞事件 不同的趋化梯度（谷氨酸，毛喉素，和NT-3）。 这 将通过分析丝状伪足活动来完成， 微管/微丝聚合/解聚和 使用组合DIC分析肌动蛋白结合蛋白凝溶胶蛋白的分布 和荧光成像方法。实现这两个目标， 提供深入了解分子事件之前的生长锥转向。 这些结果将促进我们对神经发育的理解， 并为设计指导增长的手段提供概念框架 神经损伤后再生轴突的过程

Understanding the mechanism of axonal pathfinding is a central issue in developmental neurobiology. The work outlined in this proposal is focused on the molecular and cellular mechanisms underlying the growth and navigation of nerve processes. The experimental system l will use combines in vitro study of Xenopus neurons with state-of-the-art imaging molecular manipulation techniques in the laboratory of Dr. Mu-ming Poo. The main question addressed in this proposal is how second messengers and cytoskeletaI proteins are involved in directing growth cone motility. There are two specific aims of this proposal. In AIM 1, I will determine the role of Ca2+ and cAMP as second messengers in regulating the rate and direction of neurite extension under different environmental conditions. This will be assayed by measuring [Ca2+]i at regular time intervals using the standard fura-2 ratio imaging method and measuring the rate of neurite extension by recording bright-field images using time-lapse video recording. Whether cytoplasmic Ca2+ or cAMP gradient is sufficient in inducing growth cone turning will be tested by using focal UV-activated release of Ca2+ and cAMP from caged compounds at the growth cone. Whether Ca2+ and cAMP-dependent pathways interact with each other will also be examined. In AIM 2, I will determine the early cellular events associated with the growth cone turning induced by three different chemotropic gradients (glutamate, forskolin, and NT-3). This will be accomplished by analyzing the filopodia activity, microtubule/microfilament polymerization/depolymerization and distribution of gelsolin, an actin binding protein, using combined DIC and fluorescence imaging methods. Completion of these two aims will provide insight into molecular events that precede growth cone turning. These results will both advance our understanding of neurodevelopment as well as provide conceptual framework for designing means to direct growth of regenerating axons after nerve injury.